Resveratrol Affect P53 And Related Protein Expressions In HepG2 Cell Via Caveolin-1

Objective: :Investigate the signaling mechanisms of CAV1 enhanced HepG2 cell apoptosis induced by resveratrol activate p53 .Methods: Plasmids pcDNA3.1/ NT-GFP-CAV1 (WT-CAV1 gene),CAVM1 (CAV1 gene absence 81-101 amino acids Scaffold-domain),CAVM2 (CAV1 gene absence 143-156 amino acids as lipid-binding domain)and CAV1-BLOCK-iTTM Lentiviral RNAi were transfected into HepG2 cells respectively. With 50μM resveratrol for 24 hours, use Novagen His Bind Kit isolated cells with a His tag of CAV1 protein and the interacting components. After 10% SDS-PAGE separation, Coomassie brilliant blue dye and cut the protein bands with knife,after be digested into petides by trypsin Matrix-assisted laser desorption - time of flight tandem mass spectrometry (MALDI-TOF-TOF-MS) detected of peptide sequence and contrasted with the swissprot protein database fingerprints then obtained candidate proteins interacting with CAV1. Observe the loction and expression of CAV1, p53, C-Myc, PCNA, HSP90, MDM2 and PTEN which are importance of proliferation and apoptosis by Indirect Immunofluorescence. Using western-blot detect CAV1, PTEN, pAKT, AKT, MDM2 and p53 molecules, and explore whether resveratrol activate p53 via CAV1 and PTEN-AKT signal pathway.Results:Immunoprecipitation and MALDI-TOF-TOF-MS obtained 86 candidate proteins interact with CAV1. These proteins participate in cytoskeletal protein, membrane systems and material transport protein, the nuclear transcription and translation regulatory protein, energy, protein and signaling molecules pathway. Some of these proteins consistent with artical reported, such as EGFR, NF-kB, Fas, Nitric oxide, Cyclin-G1,p53-binding protein,HSP90, PKC, etc. some of them are new candidate protein, such as SFPQ, NONO, Stathmin, Titin, Lipin-3, Glyceralde- hyde-3-phosphate dehydrogenase, Zinc finger protein, actin, ATP synthase lipid-bind -ing protein, Nucleolar GTP-binding protein 2 and so on; Indirect Immunofluorescence showed that: PCNA , MDM2 , HSP90 expression are negative with CAV1. Resveratrol can reduce PCNA, MDM2 and HSP90 expression. p53, C-Myc are Positive correlation with CAV1. Resveratrol can enhance p53, C-Myc expression. Resveratrol can significantly increase the content of PTEN in CAV1 cells. Resveratrol can stimulate p53 protein translocation to the nucleus in HepG2 cells.Futher more, Western-blot confirmed the changes of expressions of p53, MDM2 and PTEN.And showed that: resveratrol can increase CAV1, PTEN, P53 expressions, and decrease the expression of MDM2 and pAKT in HepG2 cells by Concentration-depende- nt.After stimulation of resveratrol 50μM for 24 hours, the expression of pAKT were decreased in CAV1 cells than in HepG2 cells.The situlation in CAVRNAi is opposite.Conclusion:1. CAV1 interact with a variety of proteins in tumor cell involved regulation of transcription and translation, cytoskeleton formation, membrane exchange and transfer systems, energy supply and signals.2. Resveratrol can increase p53, PTEN, C-Myc expressions while decrease HSP90, MDM2, PCNA expressions via CAV1. Resveratrol activate p53 via CAV1 and PTEN-AKT signal pathway...